Nanopore sensor for detecting molecular interactions

ABSTRACT

A nanosensor for detecting molecule characteristics includes a membrane having an opening configured to permit a charged carbon nanotube to pass but to block a molecule attached to the carbon nanotube. The opening is filled with an electrolytic solution. An electric field generator is configured to generate an electric field relative to the opening to drive the charged carbon nanotubes through the opening. A sensor circuit is coupled to the electric field generator to sense current changes due to charged carbon nanotubes passing into the opening, and to bias the electric field generator to determine a critical voltage related to a force of separation between the carbon nanotube and the molecule.

RELATED APPLICATION INFORMATION

This application is related to commonly assigned U.S. patent applicationSer. No. 13/873,854 filed on Apr. 30, 2013, incorporated herein byreference in its entirety.

BACKGROUND

1. Technical Field

The present invention relates to sensors, and more particularly to ananopore sensor and methods for detecting interaction between carbonnanotubes and proteins.

2. Description of the Related Art

Accompanied with fast-paced developments and applications ofcarbon-based nanomaterials, such as carbon nanotubes (CNTs), growingconcerns of bio-safety of these nanomaterials to a human body have ledto strategic research into nanotoxicity. It has been foundexperimentally that a CNT can pass a cell membrane and subsequentlyenter the cytoplasm and nucleus, causing cell mortality. A carbonnanotube (CNT) can be toxic to a living cell by binding to proteins andthen impairing their functionalities; however, an efficient screeningmethod that examines binding capability of a CNT to protein molecules invitro is still unavailable. At a molecular level, the nanotoxicity canresult from strong interactions between a CNT and a protein molecule,such as an insertion of a CNT into the ligand-binding site of a proteinmolecule. Consequently, a malfunction of the affected protein moleculeoccurs in cell metabolism.

On the other hand, the interaction between CNT and proteins could bebeneficial. For example, CNT-based drug molecules can be used tocompetitively interact with proteins of a virus, preventing the virusfrom attacking cells of human, animals or plants.

SUMMARY

A nanosensor for detecting molecule characteristics includes a membranehaving an opening configured to permit a charged carbon nanotube to passbut to block a molecule attached to the carbon nanotube. The opening isfilled with an electrolytic solution. An electric field generator isconfigured to generate an electric field relative to the opening todrive the charged carbon nanotubes through the opening. A sensor circuitis coupled to the electric field generator to sense current changes dueto charged carbon nanotubes passing into the opening, and to bias theelectric field generator to determine a critical voltage related to aforce of separation between the carbon nanotube and the molecule.

A nanosensor for detecting protein toxicity includes a membrane havingone or more openings, each opening being configured to permit a chargedcarbon nanotube to pass but to block a protein molecule attached to thecarbon nanotube. The opening is filled with an electrolytic solution. Anelectric field generator is configured to generate an electric fieldrelative to the opening to drive the charged carbon nanotubes throughthe opening. A sensor circuit is coupled to the electric field generatorto sense current changes due to charged carbon nanotubes passing intothe opening, and to bias the electric field generator to determine acritical voltage value for rupture between the carbon nanotube and themolecule. The critical voltage value is determined to infer interactionstrength between the carbon nanotube and the molecule.

A method for detecting molecule characteristics includes generating anelectric field across a membrane having an opening to drive chargedcarbon nanotubes through the opening, the opening being configured topermit the charged carbon nanotube to pass but to block a moleculeattached to the carbon nanotube, the opening being filled with anelectrolytic solution; sensing current changes due to charged carbonnanotubes passing into or through the opening; biasing the electricfield to measure a voltage at a point of separation between the carbonnanotube and the molecule; and correlating the voltage to measure acharacteristic of the molecule.

These and other features and advantages will become apparent from thefollowing detailed description of illustrative embodiments thereof,which is to be read in connection with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

The disclosure will provide details in the following description ofpreferred embodiments with reference to the following figures wherein:

FIG. 1 is a cross-sectional view of a nanopore-based toxicity sensor inaccordance with one embodiment;

FIG. 2 shows a cross-sectional view of a nanopore-based toxicity sensorprior to separation between the carbon nanotube and a protein inaccordance with one embodiment;

FIG. 3 shows a cross-sectional view of the nanopore-based toxicitysensor after the protein is secured in the nanopore in accordance withone embodiment;

FIG. 4 shows a cross-sectional view of the nanopore-based toxicitysensor after separation between the carbon nanotube and the protein inaccordance with one embodiment;

FIG. 5 shows a cross-sectional view of the nanopore-based toxicitysensor after further separation between the carbon nanotube and theprotein in accordance with one embodiment;

FIG. 6 is a graph showing time-dependent ionic-current signals whenelectrically driving a CNT-protein complex through a nanopore inaccordance with one embodiment;

FIG. 7 is a graph showing time-dependent ionic-current signals whenelectrically driving CNTs through a nanopore in accordance with oneembodiment;

FIG. 8 is an illustrative plot showing binding affinity versus criticalvoltage for determining separation strength between CNTs and proteins inaccordance with the present principles;

FIG. 9 is a cross-sectional view of a channel based toxicity sensor inaccordance with another embodiment;

FIG. 10 is a cross-sectional view of a multiple nanopore toxicity sensorin accordance with another embodiment;

FIG. 11 is a cross-sectional view of a multiple channel toxicity sensorin accordance with another embodiment; and

FIG. 12 is a block/flow diagram showing a method for nanosensing inaccordance with illustrative embodiments.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Studying interactions of carbon based nanotubes (CNT) to proteincomplexes remains challenging. In accordance with the presentprinciples, an ultra-sensitive (e.g., for a binding energy of a fewk_(B)T (e.g., 1-10) between CNT and protein) and high-throughput (orhigh-speed) sensor is provided that can accelerate studies in thisfield. Methods and devices are provided for detecting toxicity of a CNTto protein using a nanopore (a nanometer-sized hole in a thin membrane).In another embodiment, toxicity can be detected using multiplenanopores, using a fluid channel or multiple fluid channels. Methods anddevices for screening the toxicity of a CNT to proteins are alsoprovided by measuring the strength of a bond between the CNT and theprotein, since at the molecular level, the nanotoxicity can result fromstrong interactions between the CNT and the protein molecule. Similarmethods may be employed for other molecule, such as, e.g., singlebiological molecules, such as DNA, microRNA and etc.

It is to be understood that the present invention will be described interms of a given illustrative architecture; however, otherarchitectures, structures, materials and process features and steps maybe varied within the scope of the present invention.

It will also be understood that when an element such as a layer, region,material or device is referred to as being “on” or “over” anotherelement, it can be directly on the other element or intervening elementsmay also be present. In contrast, when an element is referred to asbeing “directly on” or “directly over” another element, there are nointervening elements present. It will also be understood that when anelement is referred to as being “connected” or “coupled” to anotherelement, it can be directly connected or coupled to the other element orintervening elements may be present. In contrast, when an element isreferred to as being “directly connected” or “directly coupled” toanother element, there are no intervening elements present.

Reference in the specification to “one embodiment” or “an embodiment” ofthe present principles, as well as other variations thereof, means thata particular feature, structure, characteristic, and so forth describedin connection with the embodiment is included in at least one embodimentof the present principles. Thus, the appearances of the phrase “in oneembodiment” or “in an embodiment”, as well any other variations,appearing in various places throughout the specification are notnecessarily all referring to the same embodiment.

It is to be appreciated that the use of any of the following “/”,“and/or”, and “at least one of”, for example, in the cases of “A/B”, “Aand/or B” and “at least one of A and B”, is intended to encompass theselection of the first listed option (A) only, or the selection of thesecond listed option (B) only, or the selection of both options (A andB). As a further example, in the cases of “A, B, and/or C” and “at leastone of A, B, and C”, such phrasing is intended to encompass theselection of the first listed option (A) only, or the selection of thesecond listed option (B) only, or the selection of the third listedoption (C) only, or the selection of the first and the second listedoptions (A and B) only, or the selection of the first and third listedoptions (A and C) only, or the selection of the second and third listedoptions (B and C) only, or the selection of all three options (A and Band C). This may be extended, as readily apparent by one of ordinaryskill in this and related arts, for as many items listed.

Referring now to the drawings in which like numerals represent the sameor similar elements and initially to FIG. 1, a cross-sectional view of acarbon nanotube (CNT)-protein complex 12 moving through a nanopore 14 isshown. The complex 12 can be prepared by mixing tested protein moleculeswith CNTs in an electrolyte solution. A CNT 30 or CNTs may bond into aligand-binding site of a protein molecule 34. Two fluidic chambers 16,18 (cis. and trans, respectively) are separated by a membrane 20 andconnected via the nanopore 14. The solid membrane 20 is formed from, forexample, SiO₂ or Si₃N₄ material, although other materials may beemployed. The fluid chambers 16, 18 are compartments that store asolution containing test molecules. The size of the nanopore ornanochannel 14 should be larger than the size of a CNT 30 and smallerthan the size of the protein molecule 34, e.g. about 3 nm in diameter. Abiasing electric field circuit or field generating circuit 23 is appliedacross the membrane 20, by inserting two electrodes 22, 24 (such as,e.g., Ag/AgCl electrodes connected to a battery 26, other electrodetypes may also be employed) into cis. and trans. chambers 16, 18,respectively.

A sensor circuit 25 is coupled to the electric field generator 23 tosense current changes due to charged carbon nanotubes passing into theopening, and to bias the electric field generator 23 (control or biasthe battery 26) through feedback 27 to reach and determine a criticalvoltage. The critical voltage, in turn, is employed to determine a forceof separation between the carbon nanotube and the molecule. A biasingvoltage may range from, e.g., about 100 mV to about 1 V.

The sensor 25 is coupled to the circuit 23 to measure changes incurrent. The sensor 25 may include known devices for accuratelymeasuring transient currents in the circuit 23, such as a patch clampamplifier. A first end 28 of the CNT 30 is functionalized with chargedchemical groups (such as a carboxyl group or amines that are negativelycharged in an electrolyte). The same end of the CNT 30 is capped so ionsdo not enter inside the CNT 30. A second end 32 of the CNT 30 isunmodified and can be used to test the toxicity of a protein molecule 34although other molecules may be employed. The second end 32 is employedto bond the CNT 30 into the ligand-binding site of the protein molecule34.

In a biasing electric field, the charged CNT 30 is electrically driventhrough the nanopore 14 as shown in FIGS. 2-5. FIG. 2 shows the CNT 30attached with the protein molecule 34 as it enters the nanopore 14. Thenanopore 14 is configured to have a size larger than a diameter of theCNT 30 and less than the size of the protein molecule 34. During thetranslocation process, as shown in FIG. 3, the protein molecule 34 istoo large to move through the nanopore 14 and is stuck inside thenanopore 14. With a biasing voltage that is larger than a critical value(V_(cr)), the CNT 30 can be further pulled through the nanopore 14 asshown in FIGS. 4 and 5. The interaction strength (thus, the potentialtoxicity) between the CNT 30 and the protein molecule 34 can be inferredfrom the critical value V_(cr) (the voltage value when the complex isruptured). The translocation process illustrated in FIGS. 2-5 can bemonitored by measuring the ionic current through the nanopore 14.Current changes will indicate when the complex 12 has entered thenanopore 14. Then a bias of the battery voltage can be performed toreach V_(cr) and rupture the complex 12. The value of V_(er) can beemployed to indicate strength and toxicity.

In one embodiment, a nanopore 14 drilled through the membrane has anhour-glass shape, with a pore “neck” 15 Å in radius (3-nm nanopore) andpore openings 25 Å in radius. A 1M KCl electrolyte was used on both cis.16 and trans. 18 sides. In one example, we adopted the atomic structureof the protein-CNT complex previously studied for CNT's toxicity to a WWdomain (1YJQ8, the smallest monomeric triple-stranded antiparallelbeta-sheet protein domain that is stable in the absence of disulfidebonds). In the complex, a (6,6)-armchair single-wall CNT (radius ˜4.1 Å)is inserted into the active site of the WW domain, which forbids theligand binding. One open end 28 of the CNT 30 is then further“functionalized” with charged atoms to mimic the carboxyl groups. Eachcharged atom has 0.5 e and the total charge of the modified CNT is 10 e.The charged CNT end 28 is capped to prevent K⁺ ions from entering theCNT. A biasing electric field, normal to the membrane surface, isapplied to drive the CNT-WW complex 12 towards the nanopore 14.

During simulations, the CNT can diffuse laterally (perpendicular to thefield direction), but atoms in the CNT are constrained within 5 Å fromthe central axis of the nanopore, permitting CNT's entry into the pore.In a biasing electric field, there exists an ionic current through thenanopore 14. The local ionic current I in the pore is calculated as

${I = {\left( {\sum\limits_{i}^{\;}{q_{i}v_{i}}} \right)/D}},$where D is the membrane thickness; q_(i) and v_(i) are the charge andthe velocity of an ion inside the pore, respectively. When the CNT-WWcomplex 12 was driven into the pore, the pore current decreased from anopen-pore current to a blockage current. Despite the fact that thecharged CNT brought its counter ions into the pore, the total number ofions inside the pore decreased. This is because ions (in a 1Melectrolyte), that were physically excluded by the complex, outnumberedcounter ions of the charged CNT. Therefore, the pore current is lesswhen the complex is inside the pore. Note that the reduction of a porecurrent indicates an entry of the complex into the pore.

Since the WW domain is larger in size than the constriction site of thenanopore, translocation of the complex is sterically prohibited. Afterthe entry (˜12 ns) of the complex, the reduced pore-current lasts forthe rest of simulation time (˜150 ns). At the same biasing voltage (0.5V), another independent simulation shows that the CNT cannot only beelectrically driven into the pore but also be electrically pulledthrough the pore, leaving the protein molecule stuck on the poresurface. During this process, a rupture between the CNT and the WWdomain occurred. The rupture process is signified by an increase of thepore current. This is expected since the blockage (by protein only) of apore current is less after the CNT exits the pore. Therefore, 0.5 V canbe considered as a critical voltage (V_(cr)), above which the rupture ofthe complex is expected. Note that the critical voltage indicates howstrongly a CNT interacts with a protein molecule.

To detect the entry of the complex into the pore, the signal of currentblockage (I_(open)−I_(b1)) needs to be larger than the noise of anopen-pore current, where I_(open) is the open pore current and I_(b1) isthe blocked pore current. We use the percentage of current blockage,i.e. (I_(open)−I_(b1))/I_(open), as a measure of signal quality. WhenV_(bias)>V_(cr), the complex is farther away from the pore constriction.When V_(bias)>V_(cr), the rupture of the complex occurs before thecomplex gets close to the pore constriction. Therefore, in both abovecases, percentages of current blockage are less than that for V_(cr).

Because of the hydrophobic interaction between the CNT and the proteinmolecule, an energy barrier needs to be overcome before the CNT can bepulled through the pore. After that, the number of contacting atoms iszero. One notable and common characteristic of these electrically drivenprocesses is that the complex moves “slowly” when distant from the porebut moves relatively faster when close to the pore. This indicates thatthe biasing electric field is stronger around the pore than far awayfrom the pore. Therefore, the electrophoretic motion of the complex isfaster when the charged part of the CNT enters the pore.

To quantify the distribution of electrostatic potentials in thesimulated system, Poisson equation was used to solve potentials fromatomic positions and charges averaged over a 4-ns simulation.Theoretically, the ionic current is proportional to(n⁺μ⁺+n⁻μ⁻)e·dV/dz·S, ignoring the contribution from gradients of ionconcentrations. Here, n⁺ and n⁻ are concentrations of cat- and an-ionsrespectively; μ⁺ and μ⁻ are mobilities of cat- and an-ions respectively;e is the charge of an electron and S is a cross-section area. Since in asteady-state the ion current is constant, dV/dz is inverselyproportional to the cross-section area. The cross-section area of ananopore is usually much smaller than that of a cis. or trans. chamber,therefore the electric field (dV/dz) inside the nanopore is much largerthan fields outside.

Referring to FIG. 6, time-dependent current signals during atranslocation/rupture event are illustratively shown in a plot ofcurrent (I) versus time (t). At the beginning, the CNT-protein complexis outside the nanopore, I_(I) is the open-pore current. At the time t₁,the complex enters the pore and the ionic current I₂ is reduced. This isdue to the fact that the complex partially blocks the nanopore. If thebiasing voltage is less than the critical value, the current remainsconstant at I₂ When the biasing voltage is larger than the criticalvalue, the CNT is further driven through the pore, and the pore is lessblocked at time t₂. Thus, the ionic current through the pore increasesto I₃. Therefore, by monitoring and/or controlling a biasing voltage, itis possible to determine the critical voltage (V_(cr)) at which arupture between the CNT and the protein molecule occurs. The ruptureforce (e.g., ˜90 pN in one case) can be estimated using q_(eff)V_(cr)/d,where q_(eff) is the effective charge of the CNT after ionic screeningin an electrolyte and d is the thickness of a solid-state membrane.

Referring to FIG. 7, time-dependent current signals are illustrativelyshown in a plot of current (I) versus time (t) for CNTs driven through ananopore without bound proteins. These events can be detected fromsignals of ionic currents shown in FIG. 7.

At the beginning, the CNT is outside the nanopore, I₁ is the open-porecurrent. At a later time, the CNT enters the pore and the ionic currentI₄ is reduced. This is due to the fact that the CNT partially blocks thenanopore, but not as much as the CNT complex in FIG. 6. Since thecross-section of CNT is smaller than a complex, the current blocked by aCNT is less (e.g., in a 1M electrolyte solution). Additionally, thetranslocation velocity of a CNT is much faster than that of a complex.Therefore, the duration time is also less, as shown in FIG. 6. It shouldbe noted that a 1M electrolyte solution is described but other ionicconcentrations may be employed.

Referring to FIG. 8, a plot of binding affinity versus critical voltageillustrates a relation that can be determined experimentally ortheoretically. The binding affinity can be inferred from the criticalvoltage (V_(cr)) at which the rupture between a CNT and a proteinmolecule occurs. This provides a high-throughput and low-cost way todetermine the interaction between a CNT and a protein molecule. Bindingaffinity may be employed as one indication of the toxicity or othercharacteristic of the protein.

Referring to FIG. 9, another embodiment shows a planar channel 40employed instead of a nanopore 14. The planar channel 40 is configuredto permit the CNT-protein complex to be separated such that the CNT 30passes into the channel 40, and the protein molecule 34 (not shown) doesnot. An electric field is applied as before with the battery 26 andelectrodes 22, 24 being employed to provide the field and to measure thecurrent changes due to the CNT-protein complex, as before. The planarchannel 40 has a channel constriction size being less than that of theprotein molecule 34.

Referring to FIG. 10, in another embodiment, multiple nanopores 114 maybe employed through a same membrane 142. The nanopores 114 function asparallel paths to process CNT-protein complexes more rapidly. A battery126 and electrodes 122, 124 are dispersed or distributed to create theelectric field.

Referring to FIG. 8, in another embodiment, instead of multiplenanopores 114, multiple channels 140 may be employed.

Referring to FIG. 9, a method for detecting molecule characteristicsusing nanopores or channels is illustratively shown. It should be notedthat, in some alternative implementations, the functions noted in theblocks may occur out of the order noted in the figures. For example, twoblocks shown in succession may, in fact, be executed substantiallyconcurrently, or the blocks may sometimes be executed in the reverseorder, depending upon the functionality involved. It will also be notedthat each block of the block diagrams and/or flowchart illustration, andcombinations of blocks in the block diagrams and/or flowchartillustration, can be implemented by special purpose hardware-basedsystems (e.g., circuitry, memory) that perform the specified functionsor acts, or combinations of special purpose hardware and computerinstructions.

In block 202, an electric field is generated across a membrane having anopening to drive charged carbon nanotubes through the opening. Theopening is configured to permit the charged carbon nanotube to pass butto block a molecule attached to the carbon nanotube in the presence ofthe electric field. The opening is filled with an electrolytic solution.In one embodiment, the opening may include one or more nanopores and/orone or more channels.

In block 204, current changes are sensed due to charged carbon nanotubespassing through the opening. The current changes are proportional to aforce of separation between the carbon nanotube and the molecule. Themolecule may include a protein. In block 205, sensing includes measuringcurrent drops and durations to determine the presence of theCNT/molecule complex in the nanopore.

In block 206, the electric field is increased/biased to reach anddetermine V_(cr). In block 207, the force of separation is determined bycomparing V_(cr) to a chart or table or computing the force from anequation. The force of separation (or binding affinity) is proportionalto toxicity, which is one of the molecule characteristics to bedetermined. The charged carbon nanotube may include a closedfunctionalized end having charged chemical groups.

Increasing the biasing value based on sensed current changes mayinclude: determining a critical voltage value at which separation occursbetween the carbon nanotube and the molecule. This V_(cr) value can becompared to a graph or other index system to determine a characteristicbased on the interaction strength. The characteristic may includebinding affinity, toxicity, etc. The toxicity measured may include thetoxicity of a CNT to a protein.

Having described preferred embodiments for a nanopore sensor fordetecting interactions between molecules (which are intended to beillustrative and not limiting), it is noted that modifications andvariations can be made by persons skilled in the art in light of theabove teachings. It is therefore to be understood that changes may bemade in the particular embodiments disclosed which are within the scopeof the invention as outlined by the appended claims. Having thusdescribed aspects of the invention, with the details and particularityrequired by the patent laws, what is claimed and desired protected byLetters Patent is set forth in the appended claims.

What is claimed is:
 1. A method for detecting molecule characteristics,comprising: generating an electric field across a membrane having anopening to drive charged carbon nanotubes through the opening, theopening being configured to permit the charged carbon nanotube to passbut to block a molecule attached to the carbon nanotube, the openingbeing filled with an electrolytic solution; sensing current changes dueto charged carbon nanotubes passing into or through the opening; biasingthe electric field to measure a voltage at a point of separation at aninterface between the carbon nanotube and the molecule, wherein thepoint of separation is located at the carbon nanotube; and correlatingthe voltage to measure a characteristic of the molecule.
 2. The methodas recited in claim 1, wherein the opening includes one of a nanoporeand a channel.
 3. The method as recited in claim 1, wherein sensingincludes measuring current drops and durations to determine when to biasthe electric field.
 4. The method as recited in claim 1, wherein themolecule includes a protein and the molecule characteristics include atoxicity.
 5. The method as recited in claim 1, wherein the chargedcarbon nanotube includes a closed functionalized end having chargedchemical groups.
 6. The method as recited in claim 1, wherein biasingthe electric field includes increasing a biasing value to determine acritical voltage value at which separation occurs between the carbonnanotube and the molecule to infer interaction strength.